ABSTRACT/SUMMARY Ryanodine receptors (RyRs) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of Ca2+ necessary to induce contraction of cardiac cells. In their intracellular environment, RyRs are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca2+) induces finely graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias), the main cause of sudden death (SD). The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in the cardiac RyR gene (hRYR2) are associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. The vast majority of CPVT mutations have been localized to three loci (hot spots) of the RyR2 protein that affect different aspects of RyR function, however, the molecular mechanism that links a mutation in the RyR2 protein and the development of tachyarrhythmias remains incompletely understood. Our general hypothesis is that CPVT mutations cause multiple forms of RyR2 dysfunction, with the severity of the phenotype determined by the hierarchy of the affected domain in the control of Ca2+ release. To test this hypothesis, we will use single RyR2 channels, isolated ventricular myocytes and whole hearts from wild-type mice and knock-in mouse models of CPVT to: (1) determine whether distinct patterns of RyR2 dysfunction emerge from each of the three hot spots altered by CPVT mutations; (2) determine whether the presumably diverse RyR2 dysfunctions elicited by mutations in each of the three hot spots converge into a preponderant cellular mechanism of aberrant electrical activity; and (3) determine if the knock-in mouse models of CPVT develop similar phenotype and respond equally to -adrenergic stimulation and -blockers. We will use an array of state-of-the-art techniques including kinetic analysis of single channel activity by laser photolysis of caged Ca2+, high-speed Ca2+ imaging with laser scanning confocal microscopy, and recording of aberrant electrical activity in whole, beating hearts. The proposed experimental design will therefore combine molecular, cellular, and whole heart studies to elucidate the molecular mechanisms of RyR-initiated tachyarrhythmias with an unprecedented level of integrative physiology.